JP2022504204A - Azithromycin and roxithromycin derivatives as aging cell death inducers - Google Patents

Abstract

The present disclosure describes the use of azithromycin, roxithromycin, and telithromycin, including derivatives, as aging cell death inducers. BrdU was used to induce aging in a human fibroblast line model. Also disclosed are methods for screening compounds for aging cell death-inducing activity. The SRB assay was used to measure cell viability via protein content. The clinically approved drugs azithromycin, roxithromycin, and telithromycin have been found to be aging cell death inducers. However, the closely related parent compound, erythromycin, did not show aging cell death-inducing activity. Azithromycin strongly induced both aerobic glycolysis and self-eating in human fibroblasts, but showed biphasic effects including mitochondrial oxygen consumption rates showing inhibitory activity at 50 μM and stimulatory activity at 100 μM. The xCELLLignence real-time assay system showed that azithromycin preferentially targets senescent cells and eliminates approximately 97% (reducing senescent cells to approximately 1/25).

Description

The present disclosure relates to an aging cell death-inducing agent, which is a compound that selectively induces cell death in aging cells.

As various organisms age with age, many genetic, phenotypic and metabolic defects accumulate. This accumulation includes signs of aging in various cell types. The overall picture of the accumulated defects is consistent with the "accumulated damage" hypothesis of aging.

Aging is a clear feature of aging with normal age. For aging, induction of CDK inhibitors such as p16-INK4A, p19-ARF, p21-WAF and p27-KIP1, and induction of SASP (senescence-associated secretory phenotype), and important lithosome enzymes. May include irreversible cell cycle cessation due to induction of (β-galactosidase) and the established aging pigment lipofustin. Interestingly, SASP is broadly inflammatory such as IL-1β and IL-6. It results in the secretion of cytokines and spreads the senescent phenotype to senescent cells "infectiously" from one cell type to another and throughout the body via chronic inflammation. Such chronic inflammation also causes cancer development, It can also promote tumor recurrence and metastasis.

Using the p16-IN4KA promoter as a transgenic probe for detecting and labeling senescent cells, several research groups have now created a mouse aging model in which senescent cells can be genetically removed in a real-time, transient manner. is doing. Although it cannot be used as an anti-aging therapy, it does provide suggestions as to whether removal of senescent cells may have therapeutic benefits to the organism. So far, great promising results have been obtained showing that genetic removal of senescent cells can actually extend healthy life expectancy and longevity.

As a result of this genetic data, many pharmaceutical companies are actively working on the development of "senescent cell death-inducing" drugs that can target senescent cells. In theory, such aging cell death inducers have the potential to undue various age-related effects. However, drug discovery is a time-consuming and costly process, requires detailed clinical trials, and is at risk of failure due to one of many possible reasons.

Therefore, it is necessary to identify compounds that are already approved for one or more treatments that also have aging cell death-inducing activity.

Brief Summary The present disclosure relates to compounds having aging cell death-inducing activity that can be used as aging cell death-inducing agents. Many FDA-approved drugs have varying degrees of aging cell death-inducing activity. Identifying such compounds and improving their selectivity for inhibition of senescent cells dramatically accelerates their availability to anti-aging drug trials. The specification describes the identification of such compounds identified after the use of controlled DNA damage as a tool to induce aging in human fibroblasts. BrdU treatment, which has a long history of use as a DNA damaging agent, can be used as an effective platform for screening compounds for aging cell death-inducing activity. More specifically, BrdU treatment makes it possible to induce aging in cultured cells with high efficiency and reproducibility.

Using BrdU treatment as a platform for identifying aging cell death-inducing activity, we have two macrolide antibiotics of the erythromycin family as clinically approved agents with efficacy as aging cell death-inducing agents. , Specifically, azithromycin and roxithromycin were identified. In terms of direct support for the high specificity of these complex interactions, the parent macrolide compound erythromycin itself lacks aging cell death-inducing activity in the screening assays disclosed herein. However, telithromycin (another macrolide) also exhibits aging cell death-inducing activity, similar to azithromycin, roxithromycin, and chemical analogs or derivatives of telithromycin. It should be noted that the terms "derivative" and "analog" are used interchangeably herein. Screening methods such as those described herein have been used to identify aging cell death inducers. In addition, aging cell death-inducing agents may be modified with one or more targeting signals to improve aging cell death-inducing activity, and others such as mitochondrial biosynthesis inhibitors to more effectively eradicate aging cells. It may be used in combination with the therapeutic agent of.

This approach can take many forms. Some embodiments include therapeutic doses of at least one aging cell death inducer selected from azithromycin, roxithromycin, telithromycin, azithromycin analogs, roxithromycin analogs, and telithromycin analogs. It may take the form of an aging cell death-inducing composition comprising. The present disclosure use the terms "derivative" and "analog" interchangeably, but their general usage may differ. For example, the plurality of embodiments may be in a pharmaceutically effective amount, in the form of a formula selected from one of the following:

In such embodiments, R1 and R2 represent functional groups, whether they are the same group or different groups, as long as at least one of R1 and R2 is one of the membrane targeting signal and the mitochondrial targeting signal. good. In other respects, R1 and R2 are hydrogen, carboxyl, alkane, cyclic alkene, alkene derivative, alkene, cyclic alkene, alkene derivative, alkyne, alkyne derivative, ketone, ketone derivative, aldehyde, aldehyde. Alkenes, carboxylic acids, carboxylic acid derivatives, ethers, ether derivatives, esters, ester derivatives, amines, amine derivatives, amides, amide derivatives, monocyclic allenes, polycyclic allenes, heteroallenes, allene derivatives , Heteroallene-based derivatives, phenols, phenol-based derivatives, benzoic acids, and benzoic acid-based derivatives. In some embodiments, at least one of R1 and R2 is a membrane targeting signal selected from palmitic acid, stearic acid, myristic acid, oleic acid, short chain fatty acids, and medium chain fatty acids. At least one of R1 and R2 is a mitochondrial targeting signal selected from tri-phenyl-phosphonium (TPP), TPP derivative, guanidinium, guanidinium derivative, and 10-N-nonylacridine orange, the senescent cell of claim 1. Death-inducing composition. In some embodiments of the aging cell death-inducing composition, at least one of R1 and R2 is 2-butene-1,4-bis-TPP; 2-chlorobenzyl-TPP; 3-methylbenzyl-TPP; 2, 4-Dichlorobenzyl-TPP; 1-naphthylmethyl-TPP; p-xylylenebis-TPP; 2-butene-1,4-bis-TPP derivative; 2-chlorobenzyl-TPP derivative; 3-methylbenzyl-TPP Derivatives; derivatives of 2,4-dichlorobenzyl-TPP; derivatives of 1-naphthylmethyl-TPP; and derivatives of p-xylylenebis-TPP.

In some embodiments, the aging cell death-inducing compound can be an azithromycin derivative or analog. For example, the aging cell death-inducing composition is expressed in the formula.

In the formula, R1 is methyl and R2 is one of the membrane targeting signal and the mitochondrial targeting signal, or R1 is the membrane targeting signal and the mitochondrial targeting signal. One of them, NH-R2, is either N (CH ₃ ) ₂ . Of course, these functional groups may differ as described elsewhere herein. Under the general usage of this technical term, a single substitution from a base compound (eg, azithromycin) is called an analog, and one or more modifications made to the base compound can be considered as a derivative. For simplicity, the terms "analog" and "derivative" are used interchangeably in this disclosure.

In some embodiments, the aging cell death-inducing compound can be a roxithromycin derivative or analog. For example, the aging cell death-inducing composition is expressed in the formula.

In the formula, is R1 O-CH ₂ -O- (CH ₂ ) ₂ -OCH ₃ and R2 is one of the membrane targeting signal and the mitochondrial targeting signal? , Or R1 is one of the membrane targeting signal and the mitochondrial targeting signal, and NH-R2 is either N (CH ₃ ) ₂ . Of course, these functional groups may differ as described elsewhere herein.

In some embodiments, the aging cell death-inducing compound can be a telithromycin derivative or analog. For example, the aging cell death-inducing composition is expressed in the formula.

In the formula, R1 can be a compound having

And R2 is one of the membrane-targeted and mitochondrial-targeted signals, or R1 is one of the membrane-targeted and mitochondrial-targeted signals and NH-R2 is N. It is either (CH ₃ ) ₂ .

In a preferred embodiment of the approach, the aging cell death-inducing composition has at least one functional group targeting signal to improve mitochondrial uptake and consequent aging cell death-inducing activity of the compound. Using the above general formula, at least one of R1 and R2 is a membrane targeting signal selected from palmitic acid, stearic acid, myristic acid, oleic acid, short chain fatty acids, and medium chain fatty acids, or 2-. Buten-1,4-bis-TPP; 2-chlorobenzyl-TPP; 3-methylbenzyl-TPP; 2,4-dichlorobenzyl-TPP; 1-naphthylmethyl-TPP; p-xylylenebis-TPP; 2-butene- Derivatives of 1,4-bis-TPP; Derivatives of 2-chlorobenzyl-TPP; Derivatives of 3-methylbenzyl-TPP; Derivatives of 2,4-dichlorobenzyl-TPP; Derivatives of 1-naphthylmethyl-TPP; and p. -One of the mitochondrial targeting signals selected from the derivatives of xylylenebis-TPP.

In some embodiments, the aging cell death-inducing composition may also include one or more additional therapeutic agents. For example, some embodiments may comprise at least one of a therapeutic amount of tetracycline, chlortetracycline, oxytetracycline, demeclocycline, metacycline, doxycycline, minocycline, and tigecycline. Some embodiments may comprise at least one of a therapeutic amount of pyruvinium, atovaquone, bedaquiline, irinotecan, sorafenib, niclosamide, stiripentol, chloroquine, and rapamycin. Also, in some embodiments, therapeutic doses of mitribocin, mitoketocin, mitoflavosine, 2-butene-1,4-bis-TPP; derivatives of 2-butene-1,4-bis-TPP; 2-chlorobenzyl- TPP; derivative of 2-chlorobenzyl-TPP; 3-methylbenzyl-TPP; derivative of 3-methylbenzyl-TPP; 2,4-dichlorobenzyl-TPP; derivative of 2,4-dichlorobenzyl-TPP; 1-naphthyl It may comprise at least one of a derivative of methyl-TPP; 1-naphthylmethyl-TPP; a derivative of p-xylylenebis-TPP; and a derivative of p-xylylenebis-TPP. Not surprisingly, the TPP derivative may be conjugated to a derivative or analog of the aging cell death-inducing compound, may act as a targeting signal, or may be present as another therapeutic compound. Some embodiments may comprise at least one of vitamin C, berberine, caffeic acid phenyl ester, silibinin, burtieridine, and melitidine.

Embodiments in the form of aging cell death-inducing compositions include, for example, cosmetics, pills, lotions, shampoos, creams, soaps, skin cleansers, shaving agents, aftershave lotions, gels, sticks, pastes, sprays, aerosols. Can be made in various forms including, powders, liquids, aqueous suspensions, aqueous solutions, foams, transdermal patches, tinctures, and steams. These forms shall be examples without limitation.

Embodiments of this approach may take the form of compositions for use in aging therapy. Such compositions include therapeutic doses of aging cell death inducers as described above. For example, aging cell death inducers are: (i) a membrane-targeted signal selected from palmitic acid, stearic acid, myristic acid, oleic acid, short-chain fatty acids, and medium-chain fatty acids; and (ii) tri-phenyl-phosphonium. (TPP), TPP derivative, guanidinium, guanidinium derivative, and azithromycin analog, loxythromycin analog, and telithromycin having at least one of the mitochondrial targeting signals selected from 10-N-nonylacridin orange. It can be one of the mycytic analogs.

In some embodiments, the composition for use in aging therapy is also a therapeutic amount of at least one of tetracycline, chlortetracycline, oxytetracycline, demeclocycline, metacycline, doxycycline, minocycline, and tigecycline. Can also include one. Some embodiments may comprise at least one of a therapeutic amount of pyruvinium, atovaquone, bedaquiline, irinotecan, sorafenib, niclosamide, stiripentol, chloroquine, and rapamycin. Also, in some embodiments, therapeutic doses of mitribocin, mitoketocin, mitoflavosine, 2-butene-1,4-bis-TPP; derivatives of 2-butene-1,4-bis-TPP; 2-chlorobenzyl- TPP; derivative of 2-chlorobenzyl-TPP; 3-methylbenzyl-TPP; derivative of 3-methylbenzyl-TPP; 2,4-dichlorobenzyl-TPP; derivative of 2,4-dichlorobenzyl-TPP; 1-naphthyl It may comprise at least one of a derivative of methyl-TPP; 1-naphthylmethyl-TPP; a derivative of p-xylylenebis-TPP; and a derivative of p-xylylenebis-TPP. In addition, some embodiments may comprise at least one of vitamin C, berberine, caffeic acid phenyl ester, silibinin, burtieridine, and melitidine.

This approach can take the form of a method for inducing cell death of senescent cells in a subject. In such an embodiment, a therapeutic amount of an aging cell death inducer is administered to the subject. The aging cell death inducer can be at least one of azithromycin, roxithromycin, telithromycin, azithromycin analog, roxithromycin analog, and telithromycin analog as described above. Derivatives or analogs may contain one or more targeting signals such as membrane targeting signals and mitochondrial targeting signals. The method may include administering one or more additional therapeutic agents. For example, a therapeutic dose of at least one of tetracycline, chlortetracycline, oxytetracycline, demeclocycline, metacycline, doxycycline, minocycline, and tigecycline can be administered with an aging cell death inducer. As another example, therapeutic doses of at least one of pyruvinium, atovaquone, bedaquiline, irinotecan, sorafenib, niclosamide, stiripentol, chloroquine, and rapamycin can be administered. As a further example, therapeutic doses of mitribocin, mitoketocin, mitoflavosin, 2-butene-1,4-bis-TPP; derivatives of 2-butene-1,4-bis-TPP; 2-chlorobenzyl-TPP; 2-chloro Derivatives of benzyl-TPP; 3-methylbenzyl-TPP; Derivatives of 3-methylbenzyl-TPP; 2,4-dichlorobenzyl-TPP; Derivatives of 2,4-dichlorobenzyl-TPP; 1-naphthylmethyl-TPP; 1 -A derivative of naphthylmethyl-TPP; p-xylylenebis-TPP; and at least one of a derivative of p-xylylenebis-TPP can be administered. As another example, a therapeutic amount of at least one of vitamin C, berberine, caffeic acid phenyl ester, silibinin, burtieridine, and melitidine can be administered. The therapeutic agent may be administered in combination with the aging cell death-inducing agent, or in some embodiments, it may be administered before or after the aging cell death-inducing agent.

Some embodiments of this approach may take the form of methods for delaying the onset of age-related diseases in a subject. Age-related diseases include atherosclerosis, arthritis, cancer, cardiovascular disease, cataracts, dementia, diabetes, hair loss, hypertension, inflammatory diseases, renal diseases, muscle atrophy, neurological diseases, degenerative arthritis, It can be at least one of osteoporosis, lung disease, disc degeneration, and alopecia. For example, age-related disorders can be neurological disorders such as mild cognitive impairment, motor neuron dysfunction, Alzheimer's disease, Parkinson's disease, and luteal degeneration. In such an embodiment, a therapeutic amount of an aging cell death-inducing agent as described herein can be administered to the subject. In some embodiments, the aging cell death inducer can be administered with another therapeutic agent as described herein. The aging cell death inducer may be administered at the onset of age-related disease, i.e., at the time of diagnosis or shortly after diagnosis. Alternatively, the aging cell death inducer may usually be administered after diagnosis and the frequency and dose can be determined using techniques known in the art. In some embodiments, the aging cell death inducer is pre-onset, especially when age-related disease is predicted or is likely to occur in the subject (eg, a genetic marker or other biological marker). Can be administered).

The approach can also take the form of a composition for use in delaying the onset of age-related diseases. For example, but not limited to, atherosclerosis, arthritis, cancer, cardiovascular disease, cataracts, dementia, diabetes, hair loss, hypertension, inflammatory disease, renal disease, muscle atrophy, It can be used to delay the onset of neurological disorders, osteoarthritis, osteoporosis, lung disorders, disc degeneration, and alopecia. In such embodiments, among therapeutic doses of azithromycin, roxithromycin, telithromycin, azithromycin analogs, roxithromycin analogs, and telithromycin analogs, as described herein. An aging cell death inducer containing at least one can be administered. In some embodiments, the aging cell death inducer can be administered with another therapeutic agent as described herein.

The approach can also take the form of a method for screening compounds for aging cell death-inducing activity. In such an embodiment, the cell population is exposed to the DNA damaging agent for the first period to produce an senescent cell population. An example of a DNA damaging agent is bromodeoxyuridine (BrdU), but other DNA damaging agents can be used without departing from this approach. At least a portion of this senescent cell population is treated with the candidate compound during the second period to form a treated cell population. Candidate compounds are compounds that are screened for aging cell death-inducing activity. This treated cell population is analyzed for at least one marker of aging cell death-inducing activity. Examples of markers of aging cell death-inducing activity include cell viability, aerobic glycolysis, self-eating, inhibitory activity, and decreased cell proliferation. For example, the self-eating LC3 protein can be measured quantitatively as described herein. The sulforhodamine B assay and the measurement of cell-induced electrical impedance are also examples of assays that can be used to analyze cell populations for aging cell death-inducing activity. The first period was mutable and was about 8 days in the embodiment below. The second period can also be changed, which is about 3 to about 5 days in the embodiments described herein. In some embodiments, BrdU may be flushed prior to the second period.

Further embodiments of this approach will be recognized by those skilled in the art by reading the detailed description below.

FIG. 1 shows a method for screening and identifying aging cell death inducers according to embodiments of this approach. FIG. 2 shows the results of decreased DNA synthesis in MRC-5 fibroblasts. FIG. 3A shows the results from the SRB assay for azithromycin in MRC-5 fibroblasts. FIG. 3B shows images of MRC-5 fibroblasts treated with 100 μM azithromycin, with and without BrdU pretreatment, respectively. FIG. 3C shows images of MRC-5 fibroblasts treated with 100 μM azithromycin, with and without BrdU pretreatment, respectively. FIG. 4 shows the results from the SRB assay for roxithromycin in MRC-5 fibroblasts. FIG. 5 shows the results of autophagy induction on 50 μM azithromycin in MRC-5 fibroblasts. FIG. 6A shows the extracellular acidification rate from metabolic flux analysis for MRC-5 cells after 72 hours of azithromycin treatment. FIG. 6B shows the extracellular acidification rate from metabolic flux analysis for MRC-5 cells after 72 hours of azithromycin treatment. FIG. 7A shows the rate of oxygen consumption from metabolic flux analysis for MRC-5 cells after 72 hours of azithromycin treatment. FIG. 7B shows the rate of oxygen consumption from metabolic flux analysis for MRC-5 cells after 72 hours of azithromycin treatment. FIG. 8 shows aging cell death-inducing activity in aging human skin cells exposed to azithromycin for 5 days after pretreatment with BrdU for 8 days to induce aging. FIG. 9A shows representative cell follow-ups obtained from xCELLLignence real-time cell health monitoring of MRC-5 cell lines comparing BrdU pretreatment, BrdU pretreatment with azithromycin, azithromycin alone, and controls. FIG. 9B summarizes the final cell index of the same strain. FIG. 10A shows the final cell index results of the MRC-5 cell line comparing BrdU pretreatment, BrdU pretreatment with roxithromycin, roxithromycin alone, and controls. FIG. 10B shows representative cell follow-ups obtained from xCELLLignence real-time cell health monitoring of MRC-5 cell lines comparing BrdU pretreatment, BrdU pretreatment with roxithromycin, roxithromycin alone, and controls. FIG. 10A shows the results of the final cell index of the MRC-5 cell line comparing BrdU pretreatment, BrdU pretreatment with telithromycin, telithromycin alone, and controls. FIG. 11B shows representative cell follow-ups obtained from xCELLLignence real-time cell health monitoring of MRC-5 cell lines comparing BrdU pretreatment, BrdU pretreatment with telithromycin, telithromycin alone, and controls.

The following description includes a mode currently planned that implements an exemplary embodiment of the approach. The following description should not be construed in a limited sense and is provided merely to illustrate the general principles of the invention.

As described herein, this approach develops a screening assay for inducing aging, as well as aging cell death-inducing activity, i.e., selective inhibition of aging cells, and induction of cell death of aging cells. With respect to its use to identify the compound provided. This approach can be used in some embodiments to identify and divert clinically approved therapeutic agents with aging cell death-inducing activity for the treatment of age-related and age-related disorders. FIG. 1 shows a screening method using this approach. In step S101, normal fibroblasts are selected. Next, in step S102, those cells are exposed to a DNA damaging agent to induce aging. For example, bromodeoxyuridine can be used as a damaging agent. These senescent cells are treated with the candidate treatment compound in step S103. Not surprisingly, a plurality of candidate treated compounds are screened using some of the senescent cells, and this step may include variants of compound concentration and treatment period. Finally, in step S104, the treated cells are analyzed for markers of aging cell death-inducing activity. Normal cells and / or untreated senescent cells can also be analyzed as controls. The aging cell death-inducing activity markers used may vary and may include, for example, cell viability, aerobic glycolysis, self-eating, inhibitory activity, and decreased cell proliferation.

Bromodeoxyuridine (5-bromo-2'-deoxyuridine), also known as BrdU, can be used to induce aging. BrdU is an analog of nucleoside thymidine commonly used to identify proliferating cells. BrdU induces controlled DNA damage and directs cells to aging with high efficiency. The BrdU assay of this approach requires normal fibroblasts to undergo long-term culture (8 days) in the presence of 100 μM BrdU to induce controlled DNA damage and aging. In an exemplary embodiment, we in a BrdU-based assay, two unrelated normal non-immortalized human fibroblast lines, MRC-5 lung cells (for screening) and BJ skin cells (for validation). )It was used. Then, homogenically matched cultures of normal fibroblasts and senescent fibroblasts can be used for drug screening to identify drugs with senescent cell death-inducing activity. Aging cell death-inducing activity can be detected using the sulforhodamine B assay, also known in the art as the SRB assay. In this assay, the amount of protein that remains associated with a tissue culture dish and is a surrogate marker of cell viability is measured. This approach can be used to rapidly screen compounds containing clinically approved drugs such as antibiotics. For example, in the embodiments described herein, the approach can be used to screen erythromycin-based members, including azithromycin and roxithromycin, among other compounds. Not surprisingly, this approach can also be used to screen for other compounds.

Mechanically, this approach directly controls and compares the responses of "normal" and "aging" fibroblasts. A drug that preferentially kills aging fibroblasts but does not kill normal fibroblasts can be considered positive for aging cell death-inducing activity. Using this approach in the embodiments described herein, we have identified two erythromycin line members, azithromycin and roxithromycin, that preferentially targeted senescent fibroblasts. Table 1 below shows the results of two concentrations of erythromycin, azithromycin, and roxithromycin, showing that azithromycin and roxithromycin have aging cell death-inducing activity at 100 μM. As well as the chemical analogs of azithromycin, roxithromycin, and telithromycin, another macrolide, telithromycin, also showed beneficial senescence cell death-inducing activity. However, erythromycin itself did not show aging cell death-inducing activity.

The exact chemical structures of some erythromycin-based members screened in the disclosed embodiments are shown below as Compounds I-IV. It should be noted that although the structures of erythromycin-based compounds have significant similarities, aging cell death-inducing activity is present in azithromycin, roxithromycin, and telithromycin.

In addition, we validated that the use of BrdU-induced DNA damage is sufficient to induce cellular senescence. FIG. 2 shows the results of decreased DNA synthesis in MRC-5 fibroblasts. BrdU treatment on the 2nd day significantly reduced DNA synthesis in MRC-5 fibroblasts by about 70% when measured using the Muse cell cycle kit. After 8 days of BrdU treatment, MRC-5 cells were positively stained for β-galactosidase, a biomarker of cellular senescence. For this data, n = 3; and ^* indicate p <0.05. The inset of FIG. 2 shows β-galactosidase positivity in BrdU-treated cells. These results demonstrate that BrdU-treated cells underwent cell cycle arrest, as evidenced by a reduction of approximately 70% in both S phase cell number and induction of β-galactosidase activity. .. From these results, it was confirmed that BrdU treatment of MRC-5 cells effectively inhibits DNA synthesis and induces β-galactosidase, both of which are characteristic of aging.

In one embodiment, azithromycin exhibited aging cell death-inducing activity in aging MRC-5 human lung fibroblasts. MRC-5 cells were pretreated with BrdU for 8 days (to induce aging), then flushed with BrdU and exposed to azithromycin for an additional 5 days. An SRB assay was then performed to determine the effect of the drug on cell viability. FIG. 3A shows the results from the SRB assay for BrdU-treated MRC-5 fibroblasts treated with 100 μM and 50 μM concentrations of azithromycin against a control (BrdU alone). 100 μM azithromycin had no effect on the viability of normal MRC-5 lung fibroblasts, but selectively killed aging MRC-5 fibroblasts. At this concentration, azithromycin effectively and selectively removed about 50% of the senescent cells after 5 days without affecting control cells. However, azithromycin was ineffective at 50 μM. These experiments were independently repeated at least 3 times with very similar results. For this data, ^** indicates p <0.01. 3B and 3C are images of MRC-5 fibroblasts treated with 100 μM azithromycin, with and without BrdU pretreatment, respectively. These images show that azithromycin had little effect on normal cells, but induced cell death in senescent cells. The upper right scale bar in FIGS. 3B and 3C represents 20 μm.

By comparison, the same concentration of roxithromycin killed aging MRC-5 fibroblasts more effectively (about 70%), but had little effect on the survival of normal MRC-5 fibroblasts. In one embodiment, MRC-5 cells were pretreated with BrdU for 8 days (to induce aging), then flushed with BrdU and exposed to roxithromycin for an additional 5 days. After 5 days of exposure, an SRB assay was performed to determine the effect of the drug on cell viability. FIG. 4 shows the results from the SRB assay for MRC-5 fibroblasts treated with roxithromycin. This data indicates that 100 μM concentration of roxithromycin had an effective and selective effect on MRC-5 cells, as more than 50% of the senescent cells were removed after 5 days. However, roxithromycin was ineffective at a concentration of 50 μM. These experiments were independently repeated at least 3 times with very similar results. In FIG. 4, ^* indicates p <0.05.

The data in FIGS. 3A and 4 show that neither azithromycin nor roxithromycin showed a significant effect on senescent cell viability at 50 μM. This indicates that the aging cell death-inducing effect is concentration-dependent. Not surprisingly, at least those with normal skill in the art will use methods known and available in the art to determine appropriate concentrations for drugs with aging cell death-inducing activity. be able to. Based on test concentrations, azithromycin toxicity showed the highest specificity for selectively targeting senescent cell phenotypes.

Further experiments were performed with MRC-5 fibroblasts to better understand the phenotypic and metabolic effects of azithromycin. MRC-5 cells were treated with 50 μM azithromycin for 72 hours. Next, autophagy was monitored by detection of autophagic LC3 protein using a kit based on Muse autophagy LC3 antibody. FIG. 5 shows these results and demonstrates that azithromycin is a promising inducer of the self-eating phenotype. ^** in FIG. 5 indicates p <0.01 of this data. As shown, azithromycin treatment resulted in a more than 3-fold increase in autophagy in MRC-5 cells.

The present inventors then measured the effect of azithromycin on aerobic glycolysis and mitochondrial metabolism using a Seahorse XFe96 metabolic flux analyzer. After treatment with azithromycin at a concentration in the range of 25 μM to 100 μM for 72 hours, metabolic flux analysis using Seahorse XFe96 was performed on MRC-5 cells, and the extracellular acidification rate (ECAR) was measured. Figures 6A and 6B show EPAR data from this metabolic flux analysis, and as can be seen, azithromycin increased aerobic glycolysis at all test concentrations. The data in FIG. 6A represent azithromycin concentrations of 100 μM, 50 μM, 25 μM, respectively, and controls (ie, vehicle alone) from top to bottom at 40 minutes. In FIG. 6B, the bar data represent controls, 100 μM, 50 μM, and 25 μM, from left to right. In this data, n = 3; ^** indicates p <0.01 and ^*** indicates p <0.001.

We also used metabolic flux analysis to assess the effect of azithromycin on oxygen consumption rate (OCR). The results shown in FIGS. 7A and 7B indicate that azithromycin has a biphasic effect on OCR in MRC-5 cells. This data was prepared by treating MRC-5 cells with metabolic flux analysis using Seahorse XFe96 after treatment with azithromycin at a concentration in the range of 25-100 μM for 72 hours. From top to bottom at 41 minutes, the line data in FIG. 7A relates to the control (ie, vehicle alone), as well as 25 μM, 50 μM, and 100 μM concentrations. It should be noted that the highest azithromycin concentration of 100 μM induced an increase in mitochondrial respiration at 54 minutes, while lower concentrations (50 μM) significantly reduced it. However, 25 μM showed no significant effect on OCR. For this data, n = 3 and ^* indicate p <0.05.

FIG. 7B shows OCR for basal respiration, proton leak, ATP-related respiration, maximal respiration, and reserve respiration capacity. From left to right, the bar data represent controls, as well as concentrations of 100 μM, 50 μM, and 25 μM. Note that the mitochondrial action of azithromycin is concentration-dependent and biphasic. At 25 μM, azithromycin showed no significant effect on OCR. However, at 50 μM, azithromycin clearly inhibited mitochondrial metabolism, especially affecting maximal and reserve respiratory capacity. In contrast, at 100 μM, azithromycin stimulated maximal respiration and the reserve respiratory capacity more than doubled. This may represent the compensatory response of the cell to overcome its mitochondrial inhibitory effect on azithromycin treatment.

The selectivity and efficacy of azithromycin were validated using normal non-immortalized BJ human fibroblasts. BJ cells were pretreated with BrdU for 8 days to induce aging, after which BrdU was washed away and exposed to azithromycin for 5 days. An SRB assay was then performed to determine the effect of azithromycin on cell viability. Azithromycin had an effective and selective effect on BJ cells since it removed more than 50% of senescent cells at 50 μM after 5 days without reducing the viability of control cells. These experiments were independently repeated at least 3 times with very similar results. The results are shown in FIG. Note that in FIG. 8, ^** indicates p <0.01. As can be seen in FIG. 8, azithromycin was more effective in BJ skin fibroblasts and showed significant senescence cell death-inducing activity at only 50 μM. Azithromycin also increased the viability of normal BJ skin fibroblasts by more than 25%. Thus, azithromycin exhibits comparable selectivity and senescence cell death-inducing activity in human fibroblasts from two different anatomical sites (lung tissue and skin).

This approach was used to test several other drug candidates using this senescence cell death induction assay system using MRC-5 or BJ fibroblasts. These compounds are listed in Table 2 above, which also shows the cell line used and the range of compound concentrations used in the test. Unfortunately, none of these other drug candidates showed specific aging cell death-inducing activity without exerting efficacy on normal fibroblast counterparts.

The erythromycin system showed no aging cell death-inducing activity as a whole, whereas azithromycin, roxithromycin, and telithromycin show specific and selective aging cell death-inducing activity. Not surprisingly, many chemical analogs and derivatives of azithromycin, roxithromycin, and telithromycin also have aging cell death-inducing activity. Traditionally, "analogs" have only one element that differs from the parent compound, and "derivatives" are chemically derived or synthesized from another. Many of the disclosed derivatives are sometimes referred to as analogs under the usual usage of this term, but these terms are used interchangeably in the present disclosure. For example, a derivative that has undergone a mono-substitution to attach a targeted signal moiety may be considered an analog. Not surprisingly, derivatives with targeting signals, such as fatty acid membrane targeting signals or TPP derivative mitochondrial targeting signals, exhibit increased mitochondrial uptake and, as a result, enhanced efficacy. This effect is significant in cells that are strongly dependent on mitochondrial biosynthesis, such as cancer stem cells and senescent cells. The following compounds V to VII are general structural formulas indicating the positions of functional groups of analogs of azithromycin, roxithromycin, and telithromycin, respectively.

Each general structure V-VII is represented by a numbered "R" and has a conjugation or substitution position called an "R group". Each R group is hydrogen, carbon, nitrogen, sulfur, oxygen, fluorine, chlorine, bromine, iodine, carboxyl, alkane, cyclic alkene, alkene derivative, alkene, cyclic alkene, alkene derivative, alkyne, alkyne derivative. , Ketones, ketone derivatives, aldehydes, aldehyde derivatives, carboxylic acids, carboxylic acid derivatives, ethers, ether derivatives, esters and ester derivatives, amines, amine derivatives, amides, amide derivatives, monocyclic or poly It can be selected from cyclic allenes, heteroallenes, allen derivatives, heteroallylen derivatives, phenols, phenolic derivatives, benzoic acids, benzoic acid derivatives, membrane targeting signals, and mitochondrial targeting signals. For a single atom such as carbon or nitrogen, it should also be understood that the valence must still be met (eg, it may require one or more additional bonds or hydrogen atoms). As used herein, the term "derivative" refers to a compound derived from a similar compound by a chemical reaction. One or more R groups of the derivative aging cell death-inducing compound can be replaced with a membrane targeting signal and / or a mitochondrial targeting signal to enhance the selectivity and efficacy of the compound for aging cells. Membrane targeting signals include, for example, palmitic acid, stearic acid, myristic acid, oleic acid, short chain fatty acids, and medium chain fatty acids. The resulting conjugate can be synthesized according to techniques known in the art to achieve chemical modification with fatty acids, such as lipidation reactions (eg, myristoylation, palmitoylation, etc.). Mitochondrial targeting signals include, for example, lipophilic cations such as tri-phenyl-phosphonium (TPP), TPP derivatives, guanidinium, guanidinium derivatives, and 10-N-nonyl acridine oranges. Non-exhaustive examples of TPP derivatives include 2-butene-1,4-bis-TPP; 2-chlorobenzyl-TPP; 3-methylbenzyl-TPP; 2,4-dichlorobenzyl-TPP; 1-naphthylmethyl. -TPP; p-xylylenebis-TPP; 2-butene-1,4-bis-TPP derivative; 2-chlorobenzyl-TPP derivative; 3-methylbenzyl-TPP derivative; 2,4-dichlorobenzyl-TPP Derivatives; 1-naphthylmethyl-TPP derivatives; and p-xylylenebis-TPP derivatives. Derivatives can be synthesized using techniques known in the art. Not surprisingly, these are non-exhaustive lists shown as examples.

In further applications of this approach, the selectivity of azithromycin for senescent cells was validated using the xCELLLignence system. Since aging cells are under the so-called aging-related secretory phenotype (SASP), which involves a dramatic increase in protein synthesis and secretion, we found that this protein measurement assay system was the aging cell of the test compound. It was assessed whether the death-inducing activity was underestimated. This xCELLurgeence assay system is protein independent and instead uses electrical impedance to continuously measure cell proliferation in real time.

9A and 9B show representative data from the xCELLLense assay for azithromycin. Representative cells tracked in FIG. 9A show that senescent cells (BrdU treated / MRC-5 fibroblasts) were effectively killed. At position 308.44 hours, from top to bottom, the curves represent BrdU alone, azithromycin alone, controls, and cells treated with BrdU and azithromycin. The normalized cell index relative to the control was significantly higher immediately after treatment, consistent with BrdU alone up to about 235 hours. FIG. 9B shows a bar graph highlighting the final cell index as mean ± standard error of mean. As can be seen, azithromycin targeted approximately 97% of aging MRC-5 cells. In contrast, normal control MRC-5 cells were only transiently affected by azithromycin and rapidly recovered via cell proliferation. The recovery exhibited by cell lines treated with azithromycin was only more than 30% above the control cell level of vehicle alone. This data confirms that azithromycin is highly efficient in targeting senescent cells preferentially, removing approximately 97% of them and reducing senescent cells by approximately 1/25. For the data shown in FIGS. 9A and 9B, ^**** indicates p <0.001. Thus, the real-time xCELLLignence assay system is a more static SRB assay and provides a more direct visualization of the potential aging cell death-inducing effect of the compound in drug screening.

The xCELLLignence assay was also used to confirm the aging cell death-inducing activity of roxithromycin and telithromycin. FIG. 10A shows the final cell index as mean ± mean standard error for BrdU-treated fibroblasts exposed to 90 μM roxithromycin-treated controls, roxithromycin-treated controls, BrdU-treated controls, respectively. MRC-5 fibroblasts were used in these assays. Compared to controls, roxithromycin treatment had the least effect on normal fibroblast viability, but targeted 82% of BrdU-treated fibroblasts to induce aging. Aging MRC-5 cells were pretreated with BrdU for 8 days to induce aging, then rinsed with BrdU and exposed to roxithromycin for an additional 5 days. These experiments were independently repeated at least 3 times with very similar results. Note that in FIG. 10A, ^* indicates p <0.01. Representative data from the xCELLLense assay for roxithromycin is shown in FIG. 10B. From top to bottom at 249 hours, the lines represent controls, BrdU alone, controls with roxithromycin (90 μM), and BrdU and roxithromycin. As can be seen, roxithromycin caused a continuous decrease in the cellular index of senescent fibroblasts. These data confirm that roxithromycin has strong aging cell death-inducing activity.

Using the same approach, we also confirmed the senescence cell death-inducing activity of telithromycin. FIG. 11A highlights the final cell index as the mean ± standard error of the mean, and FIG. 11B shows representative xCELLLignence data. The telithromycin treatment was at a concentration of 90 μM. FIG. 11A shows that telithromycin targeted 100% senescent cells and had negligible effects on normal MRC-5 cells. FIG. 11B shows controls, controls with telithromycin treatment, controls with BrdU, and BrdU and telithromycin from top to bottom at 243.5 hours. These data demonstrate that telithromycin has strong aging cell death-inducing activity.

Therefore, this approach provides an aging cell death-inducing screening method for systematically identifying compounds targeting aging phenotypic human fibroblasts. As shown in the data above and disclosed in the drawings, this approach can be used to screen for clinically approved drugs, which, of course, is not limited in scope and can be used to screen other compounds. Can also be used. These results were obtained using two well-established non-immortalized human fibroblast lines, MRC-5 and / or BJ cells. Not surprisingly, other human cells can also be used as long as induction of aging is confirmed. Fibroblasts were exposed to BrdU (DNA damaging agent) at a concentration of 100 μM for 8 days to induce cell cycle arrest and aging. Not surprisingly, other DNA damaging agents can be used without departing from this approach. Concentrations and duration of exposure are variable, but of course, induction of aging must be confirmed. After BrdU treatment and washing, fibroblasts were exposed to the test agent or compound. In the above results, the test compound exposure was 5 days. However, the time and concentration of the test compound can be varied. After drug treatment, cell attachment may be assessed by an SRB assay system using a plate reader that allows high-throughput analysis. The xCELLLense assay described above can also be used.

Using the screening method of this approach, we found that three clinically approved macrolide antibodies, azithromycin, roxithromycin, and telithromycin, have high selectivity for senescent cell death. Demonstrated to have inducing activity. In contrast, the parent compound erythromycin, which is often thought to have a similar chemical structure, did not show toxicity to senescent fibroblasts. Metabolic analysis of the chemical effects of azithromycin showed that azithromycin induced both autophagy and initiation of glycolysis. In addition, azithromycin enhanced mitochondrial activity at high doses (100 μM), but had the opposite effect at low doses (50 μM), showing a clear biphasic effect. These metabolic effects may support the highly specific aging cell death-inducing activity of azithromycin.

In summary, this approach provides a screening method for identifying compounds with aging cell death-inducing activity, such as existing clinically approved antibiotics and other drugs. This approach can be used, for example, for drug diversion as an anti-aging drug that can be used to target aging fibroblasts. This approach was used to demonstrate the selective aging cell death-inducing activity of azithromycin, roxithromycin, and telithromycin. Chemical analogs of these compounds, such as those that can be formed using the general chemical formulas above, also have aging cell death-inducing activity. In particular, derivatives containing the addition of membrane-targeted signals and / or mitochondrial targeting signals have enhanced aging cell death-inducing effects.

Thus, this approach may take the form of a method for screening compounds for aging cell death-inducing activity. The cell population may be exposed to bromodeoxyuridine (BrdU) to cause or induce aging in the cell population. One of skill in the art can determine an appropriate exposure period given the cell population and BrdU concentration, but in some embodiments the exposure may be about 8 days at a BrdU concentration of 100 μM as described above. .. The senescent cell population can be treated with a candidate compound to generate a treated cell population. The treatment period can be about 3-5 days in some embodiments, but can be changed. In some embodiments, even if a portion of the senescent cell population is treated, taking into account the untreated control moiety and, optionally, different treated compounds, different compound concentrations, and / or moieties treated at different treatment periods. good. BrdU may be rinsed off before treatment. Not surprisingly, the treatment compound, concentration, and treatment period are variable. After treatment, cell populations may be analyzed for indicators or markers of aging cell death-inducing activity. Quantitative measurements of cell viability, aerobic glycolysis, autophagy, inhibitory activity, autophagic LC3 protein, eg, using the assays described herein and / or known in the art. And may be analyzed for decreased cell proliferation. To analyze aging cell death-inducing activity, one or both of the sulforhodamine B assay and the measurement of cell-induced electrical impedance may be used as additional examples. Of course, one of ordinary skill in the art can make modifications to the assays described herein to assess the aging cell death-inducing activity of the treated compounds.

The approach may also take the form of an aging cell death-inducing composition with a therapeutic dose of at least one aging cell death-inducing agent. The aging cell death inducer can be azithromycin, roxithromycin, telithromycin, azithromycin analog, roxithromycin analog, and telithromycin analog. It should be understood that therapeutic doses are known to those of skill in the art and in the art and can be determined using available methods. In some embodiments, the aging cell death inducer is a membrane targeting signal selected from (i) palmitic acid, stearic acid, myristic acid, oleic acid, short chain fatty acids, and medium chain fatty acids; or (ii). Even if substituted with at least one targeting signal that can be any of the mitochondrial targeting signals selected from tri-phenyl-phosphonium (TPP), TPP derivatives, guanidinium, guanidinium derivatives, and 10-N-nonylacridin orange. good. Substitution of this targeting signal enhances aging cell death-inducing activity by enhancing mitochondrial uptake of aging cell death-inducing agents. For example, the R group shown in any of the above compounds IV-VI may contain a targeting signal. In some embodiments, the targeting signal can be a TPP derivative attached to an aging cell death-inducing compound at one of the substitution positions. Examples of TPP derivatives are, but are not limited to, 2-butene-1,4-bis-TPP; 2-chlorobenzyl-TPP; 3-methylbenzyl-TPP; 2,4-dichlorobenzyl-TPP; 1-naphthylmethyl-TPP; p-xylylenebis-TPP; 2-butene-1,4-bis-TPP derivative; 2-chlorobenzyl-TPP derivative; 3-methylbenzyl-TPP derivative; 2,4-dichloro Included are derivatives of benzyl-TPP; derivatives of 1-naphthylmethyl-TPP; and derivatives of p-xylylenebis-TPP. Of course, substitutions with TPP derivatives may require covalent bonds.

In some embodiments, the aging cell death-inducing agent may comprise a tetracycline compound, including tetracycline, chlortetracycline, oxytetracycline, demeclocycline, metacycline, doxycycline, minocycline, and tigecycline. These mitochondrial biosynthesis inhibitors can be used to inhibit oxidative metabolism and further amplify aging cell death-inducing activity. The data and further examples are described in International Application No. PCT / US2018 / 028587, filed April 20, 2018, the entire contents of which are incorporated herein by reference. In some embodiments, the aging cell death inducer may include glycolytic inhibitors such as pyruvinium, atovaquone, bedaquiline, irinotecan, sorafenib, niclosamide, stirpentol, chloroquine, and rapamycin. As mentioned above, senescent cells treated with senescent cell death inducers transition to the glycolytic phenotype. The introduction of glycolytic inhibitors deprives these cells of functional metabolic pathways, and therapy promotes the induction of cell death in the senescent cell population.

Under this approach, several mitochondrial biosynthesis inhibitors can be used with aging cell death inducers. Further examples of mitochondrial inhibitors include mitrivocin, oxidative and glycolytic metabolism inhibitors, repurposcins, antimitosine, mitoketocin, mitoflavosine, mitoflavin, TPP derivatives, MDIVI-1 derivatives, chloramphenicol, puromycin. Mycins and other protein synthesis inhibitors (including, for example, aminoglycosides and rapamycin analogs), antiparasitics (eg, pyruvinium pamoate, and niclosamide), chloroquin, stiripentol, caffeic acid phenyl ester (CAPE), vitamin C, 2, -Deoxy-glucose (2-DG), MCT1 inhibitors (AZD3965 and AR-C155858), D-glucosamine, quercetin, and carvegilol are included. Specific categories of mitochondrial biosynthesis inhibitor therapeutic agents are described later. For the sake of brevity, the relevant concurrency applications are incorporated herein as if they were fully indicated herein.

The first category of mitochondrial biosynthesis inhibitors is as described in International Application No. PCT / US2018 / 022403, filed March 14, 2018, the entire contents of which are incorporated herein by reference. Mitochondria. The referenced references include data on the selection of mitrivocin compounds. In general, mitribocin is a mitochondrial inhibitory compound having anticancer and often antimicrobial activity, chemotherapy sensitizing effect, radiation sensitizing effect, and photosensitizing effect, as well as anti-aging effect. These compounds bind to one (or possibly both) the large and / or small subunits of the mitochondrial ribosome and inhibit mitochondrial biosynthesis. Examples of the mitribocin group are described in the referenced application, along with general chemical structure and specific compounds, including mitribocyclins, mitribomycin, mitribosporin, and mitribophloxine. Illustrative mitrivocin is shown below as compounds VIII-XVII.

Mitoketocin is another category of mitochondrial biosynthesis inhibitors that can be used to enhance aging cell death-inducing activity. These are non-carcinogenic compounds that bind to one of ACAT1 / 2 and OXCT1 / 2 and inhibit mitochondrial ATP production. These compounds were filed on June 25, 2018 and are described in more detail in International Application No. PCT / US2018 / 039354, the entire contents of which are incorporated herein by reference. In general, mitoketocin targets mitochondrial enzymes responsible for the reuse of ketones and has anti-cancer and antibiotic properties. These compounds bind to one or both of the active catalytic sites of OXCT1 / 2 and ACAT1 / 2 and inhibit mitochondrial function.

Lipapasin and antimitosin are third category mitochondrial biosynthesis inhibitors that can be used with this approach. International Patent Application No. PCT / US2018 / 062956, filed November 29, 2018 and in its entirety is incorporated herein by reference, describes liperpacin in more detail. In general, "lipapacin" is a compound with endogenous anti-mitochondrial properties that has been chemically modified to target the compound to mitochondria. Antimitosine, a category of lipapasin, is described in more detail in International Patent Application No. PCT / US2018 / 033466, filed May 18, 2018, the entire contents of which are incorporated herein by reference. Existing antibiotics with endogenous anti-mitochondrial properties can target mitochondria and be chemically modified to inhibit mitochondrial biosynthesis. The term "antimitosin" broadly refers to an antibiotic having endogenous anti-mitochondrial properties that is chemically modified to target the antibiotic to mitochondria. Previously, the endogenous anti-mitochondrial activity of antibiotics was considered to be an unwanted side effect. In fact, some potential antibiotics have been excluded from clinical trials due to excessive anti-mitochondrial properties, and researchers have seen anti-mitochondrial activity as a potential drawback. However, under this approach, the endogenous anti-mitochondrial activity of antibiotics can be the basic invention of a whole new therapeutic agent. Antimitosin can be an antibiotic with endogenous anti-mitochondrial properties that is chemically modified with a mitochondrial targeting signal (eg, a chemical moiety). The chemical modification can be, for example, by covalent or non-covalent binding. In some embodiments, the antibiotic is one of a tetracycline family, a member of the erthyromycin family, chloramphenicol, pyruvinium pamoate, atovaquone, and bedaquiline. The mitochondrial targeting signal can be at least one compound or moiety selected from the group comprising membrane targeting signals and mitochondrial ribosome targeting signals. Examples of membrane targeting signals are short chain (eg, less than 6 carbon atoms in the chain) and medium chain (eg, 6-12 carbon atoms in the chain) fatty acids, palmitic acid, stearic acid. Includes acids, myristic acid, and oleic acid. Examples of mitochondrial ribosome targeting signals include tri-phenyl-phosphonium (TPP) and guanidinium-based moieties. TPP and guanidinium are non-toxic chemical moieties that behave functionally as mitochondrial targeting signals (MTS) in living cells. Both may be attached to the antibiotic, often via the use of carbon spacer arms or connecting chains.

Mitoflavocin and mitoflavin are fourth categories of mitochondrial biosynthesis inhibitors that can be used under this approach. These compounds are described in more detail in International Patent Application No. PCT / US2018 / 057093, filed October 23, 2018, the entire contents of which are incorporated herein by reference. Mitoflavocin is a compound that binds to flavin-containing enzymes and inhibits mitochondrial ATP production. Diphenylene iodonium chloride (DPI) is an example of mitoflavocin. Not surprisingly, mitochondria may be modified with mitochondrial targeting signals such as those described above for antimitosin. Mitochondrial mitoflavin, a derivative of riboflavin that inhibits mitochondrial function, may also be chemically modified with a mitochondrial targeting signal. For example, roseoflavin [8-demethyl-8- (dimethylamino) -riboflavin or 8-dimethylaminoriboflavin] is a natural antibacterial compound that is a derivative of riboflavin, which is involved in targeting CSCs and inhibiting mitochondrial biosynthesis. It can be chemically modified to optimize its potential. Lumichrome (7,8-dimethylaloxazine) is a fluorescent photoproduct of riboflavin degradation, which can also be chemically modified to optimize its potential for CSC targeting. .. Other common derivatives of riboflavin include alloxazine, lumiflavin, 1,5-dihydroriboflavin and 1,5-dihydroflavin. Each of these riboflavin derivatives can be chemically modified with a mitochondrial targeting signal to form mitoflavin and can be used as a mitochondrial biosynthesis inhibitor according to this approach.

A sixth category of mitochondrial biosynthesis inhibitors not only show a strong priority for uptake in cancer cells (mass cancer cells, cancer stem cells, and active cancer stem cells), but also disrupt mitochondrial biosynthesis in these cells. It is a TPP derivative compound. These TPP derivative compounds are described in more detail in International Patent Application No. PCT / US2018 / 062174, filed November 21, 2018, the entire contents of which are incorporated herein by reference. When used with respect to TPP derivatives, derivatives as known in the art are compounds that can be synthesized from a parent compound by substituting one atom with another atom or group of atoms. For example, the derivative of TPP is 2-butene-1,4-bis-TPP containing two phosphonium groups linked by butene. The derivative of 2-butene-1,4-bis-TPP may then contain the substitution of one or more phenyl groups with another compound, such as a halogen or an organic compound. For those of skill in the art, this description should be sufficient, and for the sake of brevity, this disclosure does not specify all of the possible derivatives. Other examples of TPP derivative compounds that can be used as mitochondrial biosynthesis inhibitors according to this approach are 2-butene-1,4-bis-TPP; 2-butene-1,4-bis-TPP derivatives; 2- Chlorobenzyl-TPP; Derivatives of 2-chlorobenzyl-TPP; 3-Methylbenzyl-TPP; Derivatives of 3-methylbenzyl-TPP; 2,4-Dichlorobenzyl-TPP; Derivatives of 2,4-dichlorobenzyl-TPP; 1-naphthylmethyl-TPP; derivatives of 1-naphthylmethyl-TPP; p-xylylenebis-TPP; and derivatives of p-xylylenebis-TPP are included. Not surprisingly, the above list is not an exhaustive list of TPP derivatives.

As another category of mitochondrial biosynthesis inhibitors that can be used in this approach, filed December 18, 2018, the entire content of which is incorporated herein by reference to International Patent Application No. PCT / US2018 / 0662447. It is an MDIVI-1 derivative as described. Mitochondrial division inhibitor-1 (mDIVI-1) is a small molecule that selectively and reversibly inhibits DRP1. MDIVI-1 has been shown to target DRP1 by binding and suppressing both DRP1 self-assembly to the ring-like structure around mitochondria and its GTP hydrolysis catalytic ability. This approach is a derivative of mitochondrial fission inhibitor 1 (mDIVI-1) having the general formula XVIII shown below.

Or in the form of a pharmaceutically acceptable salt thereof, where each of R1 to R8 is hydrogen, carbon, nitrogen, sulfur, oxygen, fluorine, chlorine, bromine, iodine, carboxyl, alkene, cyclic alkene, alkane. Alkenes, alkene, cyclic alkene, alkene derivatives, alkynes, alkyne derivatives, ketones, ketone derivatives, aldehydes, aldehyde derivatives, carboxylic acids, carboxylic acid derivatives, ethers, ether derivatives, esters and ester derivatives , Amines, amine derivatives, amides, amide derivatives, monocyclic or polycyclic allenes, heteroallenes, allene derivatives, heteroallene derivatives, phenols, phenolic derivatives, benzoic acids, benzoic acid derivatives, and mitochondrial targeting. It can be selected from the group consisting of signals. In some embodiments, at least one R group is a targeting signal, such as those described above.

Examples of other mitochondrial biosynthesis inhibitors that can be used to enhance the activity of aging cell death inducers under this approach include vitamin C, velverin, caffeic acid phenyl ester, silibinin, burtieridine, and melitidine. ..

Not surprisingly, aging cell death-inducing compositions have a wide range of advantageous uses. For example, the aging cell death-inducing composition can be used in aging therapies constituting the treatment of aging cells, for example by inducing cell death of aging cells and / or inhibiting the proliferation of aging cells. Aging cell death-inducing compositions include atherosclerosis, arthritis, cancer, cardiovascular disease, cataracts, dementia, diabetes, hair loss, hypertension, inflammatory diseases, renal diseases, muscle atrophy, osteoarthritis, osteoporosis. It can be used to delay the onset and / or progression of age-related diseases such as lung disease, disc degeneration, and alopecia. Neurological disorders such as mild cognitive impairment, motor neuron dysfunction, Alzheimer's disease, Parkinson's disease, and luteal degeneration can also be treated or delayed by the use of aging cell death-inducing compositions. In some cases, aging cell death-inducing compositions can be used to treat age-related diseases.

Aging cell death inducers can take many forms. For example, aging cell death inducers include cosmetics, rounds, lotions, shampoos, creams, soaps, skin cleansers, shaving agents, aftershave lotions, gels, sticks, pastes, sprays, aerosols, powders, liquids, aqueous suspensions. It can take the form of turbids, lotions, foams, aftershave patches, tinctures, and steams. For example, an aging cell death-inducing composition for treating hair loss may take the form of topical application for hair, scalp, and / or skin. Not surprisingly, those skilled in the art are familiar with selecting forms of aging cell death inducers using methods known and available in the art that do not need to be repeated here.

As mentioned above, derivatives of azithromycin, roxithromycin, and telithromycin can be used as aging cell death inducers without departing from this approach. In some embodiments, the derivative may contain one or more substitution targeting signals. Addition of a membrane-targeted or mitochondrial targeting signal to azithromycin, roxithromycin, or telithromycin significantly enhances mitochondrial uptake of the resulting conjugate, resulting in improved aging cell death-inducing activity. For example, the compounds XIX to XXI shown below represent derivatives in which one or more functional groups R are targeting signals.

First, compound XIX represents the general formula of the azithromycin derivative according to some embodiments, wherein the functional groups R1 or R2 may be the same or different, one or both of which are targeting signals. be. For example, R1 and / or R2 can be membrane-targeted signals selected from palmitic acid, stearic acid, myristic acid, oleic acid, short-chain fatty acids, and medium-chain fatty acids. (Of course, the conjugate is thought to contain a portion of the terminal where hydrogen has been removed due to binding, such as palmitate). R1 and / or R2 can be mitochondrial targeting signals selected from tri-phenyl-phosphonium (TPP), TPP derivatives, guanidinium, guanidinium derivatives, and 10-N-nonylacridine oranges. Non-exhaustive examples of TPP derivatives include 2-butene-1,4-bis-TPP; 2-chlorobenzyl-TPP; 3-methylbenzyl-TPP; 2,4-dichlorobenzyl-TPP; 1-naphthylmethyl. -TPP; p-xylylenebis-TPP; 2-butene-1,4-bis-TPP derivative; 2-chlorobenzyl-TPP derivative; 3-methylbenzyl-TPP derivative; 2,4-dichlorobenzyl-TPP Derivatives; 1-naphthylmethyl-TPP derivatives; and p-xylylenebis-TPP derivatives. In some embodiments, only one of R1 and R2 differs from the parent compound, thus the derivative is an analog. For example, R1 can be methyl and R2 can be a targeting signal. As another example, R1 can be a targeting signal and NH-R2 can be -N (CH ₃ ) ₂ . In some embodiments, one of R1 or R2 can be the targeting signal, the other of R1 and R2 being hydrogen, carboxyl, alkyne, cyclic alkyne, alkane derivative, alkene, cyclic alkene, alkene derivative. , Alkine, alkyne derivative, ketone, ketone derivative, aldehyde, aldehyde derivative, carboxylic acid, carboxylic acid derivative, ether, ether derivative, ester and ester derivative, amine, amine derivative, amide, amide derivative , Monocyclic or polycyclic allenes, heteroallenes, allen derivatives, heteroallylen derivatives, phenols, phenolic derivatives, benzoic acids, and benzoic acid derivatives can be selected from the group.

Compound XX represents the general formula of the roxithromycin derivative according to some embodiments, wherein the functional groups R1 or R2 may be the same or different, and one or both may be targeting signals. .. For example, R1 and / or R2 can be a membrane targeting signal or mitochondrial targeting signal as described above. In some embodiments, only one of R1 and R2 differs from the parent compound, thus the derivative is an analog. For example, R1 can be a methoxy such as O-CH ₂ -O- (CH ₂ ) ₂ -OCH ₃ present in roxithromycin, and R2 can be a targeting signal. As another example, R1 can be a targeting signal and NH-R2 can be N (CH ₃ ) ₂ . In some embodiments, one of R1 or R2 can be a targeting signal and the other of R1 and R2 is a hydrogen, carboxyl, alkyne, cyclic alkene, alkane derivative, alkene, cyclic alkene, alkene derivative, Alkines, alkyne derivatives, ketones, ketone derivatives, aldehydes, aldehyde derivatives, carboxylic acids, carboxylic acid derivatives, ethers, ether derivatives, esters and ester derivatives, amines, amine derivatives, amides, amide derivatives, It is selected from the group consisting of monocyclic or polycyclic allenes, heteroallenes, allen derivatives, heteroallylen derivatives, phenols, phenolic derivatives, benzoic acids, and benzoic acid derivatives.

Compound XXI represents the general formula of a telithromycin derivative, wherein the functional groups R1 or R2 may be the same or different, and one or both may be targeting signals. For example, R1 and / or R2 can be a membrane targeting signal or mitochondrial targeting signal as described above. In some embodiments, only one of R1 and R2 differs from the parent compound, thus the derivative is an analog. For example, R1 is an alkyl-aryl group, eg,

It can be present in the telithromycin carbamate ring and R2 can be a targeting signal. As another example, R1 can be a targeting signal and -NH-R2 can be -N (CH ₃ ) ₂ . In some embodiments, one of R1 or R2 can be a targeting signal and the other of R1 and R2 is a hydrogen, carboxyl, alkyne, cyclic alkene, alkane derivative, alkene, cyclic alkene, alkene derivative, Alkines, alkyne derivatives, ketones, ketone derivatives, aldehydes, aldehyde derivatives, carboxylic acids, carboxylic acid derivatives, ethers, ether derivatives, esters and ester derivatives, amines, amine derivatives, amides, amide derivatives, It is selected from the group consisting of monocyclic or polycyclic allenes, heteroallenes, allen derivatives, heteroallylen derivatives, phenols, phenolic derivatives, benzoic acids, and benzoic acid derivatives.

In the following example, compound XXII represents an azithromycin derivative, where R1 is methyl and R2 is a fatty acid membrane targeting signal. In this example, R2 is a fatty acid moiety derived from the membrane targeting signal myristic acid. As a matter of course, compound XXII can be formed, for example, by myrostoylation, a lipidation technique known in the art. This example of an aging cell death-inducing compound increased mitochondrial uptake more than azithromycin and, as a result, enhanced aging cell death-inducing activity. Compound XXII is an example of an azithromycin derivative of this approach having aging cell death-inducing activity. Not surprisingly, many other derivatives with aging cell death-inducing activity can be made under this approach.

Later, the materials and methods used for the experiments and data disclosed herein are described. Not surprisingly, these materials and methods are exemplary and one of ordinary skill in the art can make changes without departing from this approach. MRC-5 (ATCC® CCL-171) human lung fibroblasts and BJ (ATCC® CRL2522) human skin fibroblasts were purchased from ATCC (American Type Culture Collection). Gibco brand cell culture medium (MEM) was purchased from Life Technologies. Bromodeoxyuridine, azithromycin, roxithromycin and erythromycin were purchased from Sigma-Aldrich. Azithromycin (from Pfizer) is FDA approved. Roxithromycin (from GSK and Sandoz) is not available in the United States, but is clinically approved in New Zealand, Australia and Israel.

MRC-5 cells or BJ cells were seeded in 24-well plates. The next day, half of the plate was treated with 100 μM BrdU and the control wells were treated with vehicle alone (DMSO) and incubated at 37 ° C. for 8 days in a 5% CO ₂ humidified atmosphere. After 8 days of BrdU treatment, cells were treated with various test compounds or drugs (eg, azithromycin, roxithromycin, telithromycin, erythromycin, etc.) for an additional 3-5 days. BrdU should be rinsed before drug treatment.

Sulforhodamine B Assay: After plate incubation, cell viability was measured by the Sulforhodamine B Assay (SRB). This assay is based on measuring the protein content of cells. Cells were fixed at 4 ° C. for 1 hour with 10% trichloroacetic acid (TCA) and dried overnight at room temperature. The plates were then incubated with SRB for 30 minutes, washed twice with 1% acetic acid and air dried for at least 1 hour. Finally, the dye bound to the protein was dissolved in a 10 mM Tris, pH 8.8 solution and read at 540 nm using a plate reader.

Self-eating and cell cycle analysis: Self-eating (using the Muse ™ self-eating LC3 antibody-based kit, Merck Millipore) and cell cycle (Muse® cell cycle kit, Merck Millipore) tests according to the manufacturer's instructions. went.

Beta-Gal staining: β-galactosidase staining of BrdU-treated MRC-5 cells was performed with an aging β-galactosidase staining kit (# 9860, Cell Signaling Technology Inc.) according to the manufacturer's protocol.

Seahose XFe96 Metabolic Flux Analysis: MCF7 cell extracellular acidification rate (ECAR) and real-time oxygen consumption rate (OCR) were determined using a Seahorse extracellular flux (XF96) analyzer (Seahorse Bioscience, Mass., USA). .. MRC-5 cells were maintained in MEM supplemented with 10% FBS (fetal bovine serum), 2 mM GlutaMAX, and 1% Pen-Strep. 40,000 cells per well were seeded on XF96 well cell culture plates and incubated overnight at 37 ° C. in a 5% CO2 humidified atmosphere. The next day, cells were treated with azithromycin for 72 hours. Prior to the test, the plates were washed with a preheated XF assay medium (10 mM glucose, 1 mM pyruvate was added to the XF assay medium for OCR measurement and adjusted to pH 7.4). The cells were then maintained in a non-CO2 incubator at 37 ° C. for 1 hour in a 175 μL / well XF assay medium. During incubation, 25 μL of 80 mM glucose, 9 μM oligomycin, 1M 2-deoxyglucose (for EPAR measurement) and 25 μL of 10 μM oligomycin, 9 μM FCCP, 10 μM rotenone, 10 μM antimycin A (for OCR measurement) in the XF assay medium. Was added to the injection port of the XFe-96 sensor cartridge. During the test, the device injected these inhibitors into the wells at a given time point and EPAR / OCR was continuously measured. EPAR and OCR measurements were normalized by protein content (sulforhodamine B assay). Data sets were analyzed by XFe-96 software using one-way ANOVA and Student's t-test calculations. For the results disclosed herein, all tests were performed in at least 3 iterations.

xCELLConnection assay system: xCELLurgeenceRTCA system (ACEA Biosciences Inc.). Briefly, MRC-5 pulmonary fibroblasts (vehicle alone and / or treated with 100 μM BrdU) are seeded in each well and azithromycin is used with RTCA (real-time cell analysis) by measuring the electrical impedance induced by the cells. It was used to evaluate the effectiveness of. This approach allows the induction of cellular responses and the quantification of rate. The test was repeated several times independently, using 4 iterations of the sample for each condition.

Statistical analysis: Statistical significance was determined using Student's t-test and values less than 0.05 were considered significant. Data are shown as mean ± SEM.

The technical terms used herein are solely for the purpose of describing particular embodiments and are not intended to limit this approach. As used herein, the singular forms "one (a)", "one (an)", and "the" are plural unless the context clearly indicates otherwise. Also included. In addition, the term "comprises" and / or "comprising", when used herein, specifies the presence of the features, integers, steps, operations, elements, and / or components described. It will be appreciated that it does not preclude the existence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

The present invention may also be embodied in other particular forms without departing from its gist or its essential features. Accordingly, the embodiments should be considered in all respects to be exemplary and not limited, and the scope of the invention is set forth by the claims of the present application rather than by the above description, and thus the claims. Any change within the meaning of the range and within its uniformity shall be included therein.

Claims (50)

The following formula

In the formula, R1 and R2 may be the same or different, membrane targeting signal, mitochondrial targeting signal, hydrogen, carboxyl, alkyne, cyclic alkyne, alkane derivative, alkene, cyclic alkene, alkene system. Derivatives, alkynes, alkyne derivatives, ketones, ketone derivatives, aldehydes, aldehyde derivatives, carboxylic acids, carboxylic acid derivatives, ethers, ether derivatives, esters, ester derivatives, amines, amine derivatives, amides, amides Selected from derivatives, monocyclic allenes, polycyclic allenes, heteroallenes, allene derivatives, heteroallene derivatives, phenols, phenolic derivatives, benzoic acids, and benzoic acid derivatives; and at least one of R1 and R2 is a membrane. An aging cell death-inducing composition comprising a pharmaceutically effective amount of a compound having a formula selected from the targeting signal and one of the mitochondrial targeting signals. The aging cell death-inducing composition according to claim 1, wherein at least one of R1 and R2 is a membrane-targeted signal selected from palmitic acid, stearic acid, myristic acid, oleic acid, short-chain fatty acids, and medium-chain fatty acids. thing. 1. Aging cell death-inducing composition. At least one of R1 and R2 is 2-butene-1,4-bis-TPP; 2-chlorobenzyl-TPP; 3-methylbenzyl-TPP; 2,4-dichlorobenzyl-TPP; 1-naphthylmethyl-TPP; p-xylylenebis-TPP; 2-butene-1,4-bis-TPP derivative; 2-chlorobenzyl-TPP derivative; 3-methylbenzyl-TPP derivative; 2,4-dichlorobenzyl-TPP derivative; 1 The aging cell death-inducing composition according to claim 3, which is a TPP derivative selected from a derivative of -naphthylmethyl-TPP; and a derivative of p-xylylenebis-TPP. The compound is of the formula

In the formula, R1 is methyl and R2 is one of the membrane-targeted and mitochondrial-targeted signals, or R1 is one of the membrane-targeted and mitochondrial-targeted signals. , NH-R2 is any of N (CH ₃ ) ₂ , the aging cell death-inducing composition according to claim 1. The compound is of the formula

In the formula, R1 is O-CH ₂ -O- (CH ₂ ) ₂ -OCH ₃ , R2 is one of the membrane-targeted and mitochondrial-targeted signals, or R1 is membrane-targeted. The aging cell death-inducing composition according to claim 1, wherein one of the signal and the mitochondrial targeting signal, NH-R2 is either N (CH ₃ ) ₂ . The compound is of the formula

In the formula, R1 is

And R2 is one of the membrane-targeted and mitochondrial-targeted signals, or R1 is one of the membrane-targeted and mitochondrial-targeted signals and NH-R2 is N. (CH ₃ ) The aging cell death-inducing composition according to claim 1, which is either ₂ . At least one of R1 and R2 is a membrane-targeted signal selected from palmitic acid, stearic acid, myristic acid, oleic acid, short-chain fatty acids, and medium-chain fatty acids, or at least one of R1 and R2 is 2. -Buten-1,4-bis-TPP; 2-chlorobenzyl-TPP; 3-methylbenzyl-TPP; 2,4-dichlorobenzyl-TPP; 1-naphthylmethyl-TPP; p-xylylenebis-TPP; 2-butene -1,4-Bis-TPP derivative; 2-chlorobenzyl-TPP derivative; 3-methylbenzyl-TPP derivative; 2,4-dichlorobenzyl-TPP derivative; 1-naphthylmethyl-TPP derivative; and The aging cell death-inducing composition according to any one of claims 5 to 7, which is any of the mitochondrial targeting signals selected from the derivative of p-xylylenebis-TPP. Aging cell death-inducing composition comprising a therapeutic amount of at least one aging cell death-inducing agent selected from azithromycin, roxithromycin, telithromycin, azithromycin analog, roxithromycin analog, and telithromycin analog. thing. The aging cell death inducer is (i) a membrane-targeted signal selected from palmitic acid, stearic acid, myristic acid, oleic acid, short-chain fatty acids, and medium-chain fatty acids; and (ii) tri-phenyl-phosphonium (ii). TPP), TPP derivatives, guanidinium, guanidinium derivatives, and azithromycin analogs, loxythromycin analogs with targeting signals, including at least one of the mitochondrial targeting signals selected from 10-N-nonylacridin orange. The aging cell death-inducing composition according to claim 9, which comprises, and one of the terisuromycin analogs. The targeting signal is 2-butene-1,4-bis-TPP; 2-chlorobenzyl-TPP; 3-methylbenzyl-TPP; 2,4-dichlorobenzyl-TPP; 1-naphthylmethyl-TPP; p- Xylylenebis-TPP; 2-butene-1,4-bis-TPP derivative; 2-chlorobenzyl-TPP derivative; 3-methylbenzyl-TPP derivative; 2,4-dichlorobenzyl-TPP derivative; 1-naphthyl The aging cell death-inducing composition according to claim 10, which is a TPP derivative selected from a derivative of methyl-TPP; and a derivative of p-xylylenebis-TPP. The aging according to any one of claims 9 to 11, further comprising a therapeutic amount of at least one of tetracycline, chlortetracycline, oxytetracycline, demeclocycline, metacycline, doxycycline, minocycline, and tigecycline. Cell death-inducing composition. The aging cell death-inducing composition according to any one of claims 9 to 11, further comprising a therapeutic amount of at least one of pyrvinium, atovaquone, bedaquiline, irinotecan, sorafenib, niclosamide, stiripentol, chloroquine, and rapamycin. thing. Therapeutic doses of mitribocin, mitoketocin, mitoflavosine, 2-butene-1,4-bis-TPP; derivatives of 2-butene-1,4-bis-TPP; 2-chlorobenzyl-TPP; 2-chlorobenzyl-TPP Derivatives; 3-Methylbenzyl-TPP; Derivatives of 3-Methylbenzyl-TPP; 2,4-Dichlorobenzyl-TPP; Derivatives of 2,4-dichlorobenzyl-TPP; 1-naphthylmethyl-TPP; 1-naphthylmethyl- The aging cell death-inducing composition according to any one of claims 9 to 11, further comprising at least one of a derivative of TPP; p-xylylenebis-TPP; and a derivative of p-xylylenebis-TPP. The aging cell death-inducing composition according to any one of claims 9 to 14, further comprising at least one of vitamin C, berberine, caffeic acid phenyl ester, silibinin, burtieridin, and melitidine. Cosmetics, pills, lotions, shampoos, creams, soaps, skin cleansers, shaving agents, aftershave lotions, gels, sticks, pastes, sprays, aerosols, powders, liquids, aqueous suspensions, aqueous solutions, foams, transdermal The aging cell death-inducing composition according to claim 9, which is in the form of at least one of a patch, a tincture, and a steam. Used in aging therapy, including therapeutic doses of aging cell death-inducing agents comprising at least one of azithromycin, roxithromycin, telithromycin, azithromycin analog, roxithromycin analog, and telithromycin analog. Composition for The aging cell death inducer is (i) a membrane-targeted signal selected from palmitic acid, stearic acid, myristic acid, oleic acid, short-chain fatty acids, and medium-chain fatty acids; and (ii) tri-phenyl-phosphonium (TPP). ), TPP derivative, guanidinium, guanidinium derivative, and at least one of the mitochondrial targeting signals selected from 10-N-nonylacridin orange, azithromycin analog, loxythromycin analog, and telithromycin analog. 17. The composition of claim 17, comprising one of the bodies. The composition according to any one of claims 17-18, further comprising a therapeutic amount of at least one of tetracycline, chlortetracycline, oxytetracycline, demeclocycline, metacycline, doxycycline, minocycline, and tigecycline. .. The composition according to any one of claims 17-18, further comprising a therapeutic amount of at least one of pyrvinium, atovaquone, bedaquiline, irinotecan, sorafenib, niclosamide, stiripentol, chloroquine, and rapamycin. Therapeutic doses of mitrivosin, mitoketocin, mitoflavosine, 2-butene-1,4-bis-TPP; 2-butene-1,4-bis-TPP derivative; 2-chlorobenzyl-TPP; 2-chlorobenzyl-TPP derivative 3-Methylbenzyl-TPP; Derivatives of 3-Methylbenzyl-TPP; 2,4-Dichlorobenzyl-TPP; Derivatives of 2,4-dichlorobenzyl-TPP; 1-naphthylmethyl-TPP; 1-naphthylmethyl-TPP The composition according to any one of claims 17 to 18, further comprising at least one of a derivative of p-xylylenebis-TPP; and a derivative of p-xylylenebis-TPP. The composition according to any one of claims 17-18, further comprising at least one of vitamin C, berberine, caffeic acid phenyl ester, silibinin, burtieridin, and melitidine. A method for inducing senile cell death in a subject of a therapeutic amount of azithromycin, roxithromycin, telithromycin, azithromycin analog, roxithromycin analog, and telithromycin analog. A method comprising administering an aging cell death-inducing agent comprising at least one of them. The aging cell death inducer is one of azithromycin analogs, loxythromycin analogs, and terrislomycin analogs, as well as (i) palmitic acid, stearic acid, myristic acid, oleic acid, short chain fatty acids. And membrane targeting signals selected from medium chain fatty acids; and (ii) mitochondrial targeting signals selected from tri-phenyl-phosphonium (TPP), TPP derivatives, guanidinium, guanidinium derivatives, and 10-N-nonylacridin orange. 23. The method of claim 23, comprising at least one of. 23. The method of claim 23, further comprising administering a therapeutic amount of at least one of tetracycline, chlortetracycline, oxytetracycline, demeclocycline, metacycline, doxycycline, minocycline, and tigecycline. 23. The method of claim 23, further comprising administering a therapeutic amount of at least one of pyrvinium, atovaquone, bedaquiline, irinotecan, sorafenib, niclosamide, stiripentol, chloroquine, and rapamycin. Therapeutic doses of mitribocin, mitoketocin, mitoflavosine, 2-butene-1,4-bis-TPP; 2-butene-1,4-bis-TPP derivative; 2-chlorobenzyl-TPP; 2-chlorobenzyl-TPP derivative 3-Methylbenzyl-TPP; Derivative of 3-Methylbenzyl-TPP; 2,4-Dichlorobenzyl-TPP; Derivative of 2,4-dichlorobenzyl-TPP; 1-naphthylmethyl-TPP; 1-naphthylmethyl-TPP 23. The method of claim 23, further comprising administering at least one of a derivative of p-xylylenebis-TPP; and a derivative of p-xylylenebis-TPP. 23. The method of claim 23, further comprising administering at least one of vitamin C, berberine, caffeic acid phenyl ester, silibinin, burtieridine, and melitidine. A method for delaying the onset of age-related diseases in a subject, wherein the subject has therapeutic doses of azithromycin, roxithromycin, telithromycin, azithromycin analog, roxithromycin analog, and telithromycin. A method comprising administering an aging cell death-inducing agent comprising at least one of the analogs. The age-related diseases include atherosclerosis, arthritis, cancer, cardiovascular disease, cataract, dementia, diabetes, hair loss, hypertension, inflammatory disease, renal disease, muscle atrophy, neurological disease, degenerative arthritis, 29. The method of claim 29, comprising at least one of osteoporosis, lung disease, disc degeneration, and alopecia. 30. The method of claim 30, wherein the neurological disorder comprises at least one of mild cognitive impairment, motor neuron dysfunction, Alzheimer's disease, Parkinson's disease, and luteal degeneration. 29. The method of claim 29, wherein the aging cell death-inducing composition comprises at least one of an oral composition, a topical composition, and an intravenous composition. The method according to any one of claims 29 to 32, wherein the aging cell death inducer comprises the compound according to any one of claims 1 to 8. One of claims 29-33, further comprising administering a therapeutic amount of at least one of tetracycline, chlortetracycline, oxytetracycline, demeclocycline, metacycline, doxycycline, minocycline, and tigecycline. The method described. The method of any one of claims 29-33, further comprising administering a therapeutic amount of at least one of pyrvinium, atovaquone, bedaquiline, irinotecan, sorafenib, niclosamide, stiripentol, chloroquine, and rapamycin. Therapeutic doses of mitribocin, mitoketocin, mitoflavosine, 2-butene-1,4-bis-TPP; 2-butene-1,4-bis-TPP derivative; 2-chlorobenzyl-TPP; 2-chlorobenzyl-TPP derivative 3-Methylbenzyl-TPP; Derivative of 3-Methylbenzyl-TPP; 2,4-Dichlorobenzyl-TPP; Derivative of 2,4-dichlorobenzyl-TPP; 1-naphthylmethyl-TPP; 1-naphthylmethyl-TPP The method of any one of claims 29-33, further comprising administering at least one of a derivative of p-xylylenebis-TPP; and a derivative of p-xylylenebis-TPP. The method of any one of claims 29-36, further comprising administering at least one of vitamin C, berberine, caffeic acid phenyl ester, silibinin, burtieridine, and melitidine. A composition for use in the delayed onset of age-related diseases of therapeutic doses of azithromycin, roxithromycin, telithromycin, azithromycin analogs, roxithromycin analogs, and telithromycin analogs. A composition comprising an aging cell death inducer comprising at least one of them. The age-related diseases include atherosclerosis, arthritis, cancer, cardiovascular disease, cataract, dementia, diabetes, hair loss, hypertension, inflammatory disease, renal disease, muscle atrophy, neurological disease, degenerative arthritis, 38. The composition of claim 38, comprising at least one of osteoporosis, lung disease, disc degeneration, and alopecia. The aging cell death-inducing agent is a therapeutic amount of tetracycline, chlorotetracycline, oxytetracycline, demecrocycline, metacycline, docycycline, minocycline, tigecycline, pyruvinium, atobacon, vedakirin, irinotecan, sorafenib, niclosamide, stylapentol, chloroquinol. , Mitrivocin, mitoketocin, mitoflavosine, 2-butene-1,4-bis-TPP; derivative of 2-butene-1,4-bis-TPP; 2-chlorobenzyl-TPP; derivative of 2-chlorobenzyl-TPP; 3 -Methylbenzyl-TPP; Derivatives of 3-methylbenzyl-TPP; 2,4-Dichlorobenzyl-TPP; Derivatives of 2,4-dichlorobenzyl-TP P; 1-naphthylmethyl-TPP; 1-naphthylmethyl-TPP Derivatives; p-xylylenebis-TPP; derivatives of p-xylylenebis-TPP; any of claims 38 and 39 further comprising at least one of vitamin C, velverin, caffeic acid phenyl ester, siribinin, butyleridine, and melitidine. The composition according to paragraph 1. The composition according to claim 28, wherein the aging cell death inducer comprises the compound according to any one of claims 1 to 8. A method for screening compounds for aging cell death-inducing activity.
Exposing the cell population to a DNA damaging agent for the first period to produce an senescent cell population;
Treating at least a portion of the senescent cell population with a candidate compound for a second period to form a treated cell population;
A method comprising analyzing the treated cell population for at least one marker of aging cell death-inducing activity. 42. The method of claim 42, wherein the DNA damaging agent comprises bromodeoxyuridine (BrdU). 42. The method of claim 42, wherein the at least one marker of aging cell death-inducing activity comprises at least one of cell viability, aerobic glycolysis, phagocytosis, inhibitory activity, and decreased cell proliferation. 42. The method of claim 42, wherein the at least one marker of aging cell death-inducing activity comprises a quantitative measurement of an autophagic LC3 protein. 42. The method of claim 42, wherein the first period is about 8 days. 42. The method of claim 42, wherein the second period is from about 3 days to about 5 days. 42. The method of claim 42, wherein BrdU is washed away from the senescent cell population prior to a second period. 42. The method of claim 42, further comprising analyzing at least a portion of the senescent cell population for at least one marker of senescent cell death-inducing activity. Analyzing the treated cell population for at least one marker of aging cell death-inducing activity comprises at least one of performing a sulforhodamine B assay and measuring cell-induced electrical impedance, claim 42. The method described.

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